The focus of this proposal is the development of adeno-associated virus (AAV) vector targeting strategies for therapeutic gene delivery. Recombinant AAV represents an appealing vector system for human gene therapy due to its lack of pathogenicity, long-term transduction potential, and it's ability to transduce quiescent cells. However, regardless of its potential promise, the AAV vector system still faces certain inherent problems. One such problem relates to the endogenous tissue tropism of AAV and the limitations that this places on the development of AAV vectors for specific gene therapy applications. The central hypothesis of this proposal is that by characterizing and modifying the AAV capsid proteins we will not only increase our understanding of viral tissue tropism, entry, immunoreactivity, and particle assembly, but also be able to develop AAV-based vector systems for the targeted delivery of genes to specific cell populations. First, this project will examine our ability to target AAV vectors to specific cellular receptors by incorporating ligand polypeptides into the vector system. Second, by characterizing AAV capsid functional domains we will further define the process of AAV infection and gain a better understanding of the biological properties of these reagents that will ultimately expedite their introduction into the clinical arena. As such, this proposal has three aims. First, the characterization of functional domains within the AAV capsid proteins and the identification of sites that tolerate peptide insertion. These studies will also allow the definition of capsid regions essential for the viral life cycle, including regions of the capsid involved in binding to cellular receptors, entry and trafficking of viral particles in host cells, packaging of viral DNA, and particle assembly. Second, the development of AAV vectors with altered cell/tissue tropism by incorporating ligand polypeptides into the vector system. Third, the determination of the ability of AAV vectors with modified capsid proteins or attached targeting ligands to direct gene transduction to specific target cell populations relevant to effective nuclear entry and expression of AAV vector transgenes. We will also examine the intracellular consequences of entry via an alternative receptor, in regard to altered trafficking and induction of host innate immune responses, and we will examine the in vivo targeting and biodistribution of AAV vectors with altered tropism.